1. Field of the Invention
The present invention relates to the use of surface display technology as a uniform catalyst support for synthetic catalysts. Catalyst supports of the invention include uniform layers of catalyst on their surface, allowing catalysis with properties of homogeneous catalysts, while still allowing efficient separation and recycling of the catalyst. Although a variety of biological supports are taught herein, yeast cell-supported catalysts are particularly preferred.
2. Background
A. Cell Surface Displays
A number of microorganisms and virions may be engineered so that proteins are genetically displayed on their surface. The yeast Saccharomyces cerevisiae is often used for this process. A number of methods, structures, and applications of yeast cell-surface display have been reported. Kondo, A., and Ueda, M., “Yeast Cell-Surface Display—Applications of Molecular Display,” Appl. Microbiol. Biotechnol. 64:28-40 (2004); Ueda, M. & Tanaka, A., “Cell Surface Engineering of Yeast: Construction of Arming Yeast with Biocatalyst” J. Biosci. and Bioeng. 90(2): 125-136 (2000).
Boder and Wittrup report that S. cerevisiae is ideal for cell surface display of proteins, because S. cerevisiae possesses both secretory machinery and protein folding mechanisms that are similar to those of mammalian cells. (Boder, E. T. & Wittrup, D. K., “Yeast Surface Display for Screening Combinatorial Polypeptide Libraries,” Nature Biotech. 15:553-557 (1997)). Other benefits include large numbers of surface fusions per cell and the ability to test the cells using flow cytometry. (Boder, et al.). Yeast cells may display proteins of differing stability and expression level, as reported by Park, et al., “Limitations of Yeast Surface Display in Engineering Proteins of High Thermostability,” PEDS, 19(5): 211-217 (2006).
Yeast cell surface display technology is further reported in U.S. Pat. No. 6,423,538, to Wittrup, et al., “Yeast Cell Surface Display of Proteins and Uses Thereof.” Wittrup, et al., reports a genetic method for anchoring polypeptides to a yeast cell wall. U.S. Pat. No. 6,300,065, to Kieke, et al., “Yeast Cell Surface Display of Proteins and Uses Thereof,” reports a method for the fusion of the N-terminus of a “polypeptide of interest” to the C-terminus of the yeast Aga2p cell wall protein.
Kieke's method may purportedly be used for anchoring an scFv antibody fragment to exterior of the yeast cell wall. Yeast cell surface display has also been reported for use in antibody epitope mapping. Chao, et al., “Fine Epitope Mapping of Anti-Epidermal Growth Factor Receptor Antibodies Through Random Mutagenesis and Yeast Surface Display,” J. Mol. Biol., 342(2): 539-550 (2004); see also Colby, et al., “Development of a Human Light Chain Variable Domain (VL) Intracellular Antibody Specific for the Amino Terminus of Huntingtin via Yeast Surface Display,” J. Molec. Biol. 342(3): 901-912 (2004); Min Li, “Applications of Display Technology in Protein Analysis,” Nature Biotech. 18:1251-1256 (2000); Feldhaus, M. J. & Siegel, R. W., “Yeast Display of Antibody Fragments: A Discovery and Characterization Platform,” J. Immunol. Methods 290(1-2): 69-80 (2004); Weaver-Feldhaus, et al., “Directed Evolution for the Development of Conformation-Specific Affinity Reagents using Yeast Display” 18(11): 527-536 (2005); Little, et al., “Bacterial Surface Presentation of Proteins and Peptides: An Alternative to Phage Technology?” Trends in Biotechnology 11 (1993).
Cell surface display has also reportedly been used for absorption of environmental metals. Wernerus, H. & Stahl, S., “Biotechnological Applications for Surface-Engineered Bacteria” Biotechnol. Appl. Biochem. 40:209-228 (2004). Cell surface display has also reportedly been used to bind eukaryotic cell display libraries to solid surfaces. Peelle, et al., U.S. Pat. Appl'n No. U.S.2006/0003387, “Cell Display Libraries”; Andres, et al., “Immobilization of Saccharomyces cerevisiae Cells to Protein G-Sepharose by Cell Wall Engineering” J. Mol. Microbiol. & Biotech. 5(3) 161-166 (2003). Expression of the ZZ domain from Staphylococcus aureus, which binds to the Fc part of immunoglobulin G (IgG) has also been reported. Shimojyo, et al., “Preparation of Yeast Strains Displaying IgG Binding Domain ZZ and Enhanced green Fluorescent Protein for Novel Antigen Detection Systems” J. Biosci & Bioeng. 96(5):493-495 (2003).
Kondo reports that yeast cell surface display has been used for biocatalysis; however, Kondo's biocatalysts have a number of disadvantages that hinder their general applicability. For example, the biocatalysts are bound directly to the surface of the yeast cell. Such catalysts may not be interchanged for other catalysts as the need arises, and familial yeast lines are limited to a single biocatalyst configuration. Furthermore, biocatalyst that is adsorbed to an unwanted target is difficult or impossible to remove or separate from a cell without irrevocably damaging the cell. Biocatalysts are also traditionally limited to those catalysts that may be expressed directly on the cell surface, such as protein molecules or enzymes.
Cell surface display using microorganisms other than yeast has also been reported. For example, Wang, et al., “Specific Adhesion to Cellulose and Hydrolysis of Organophosphate Nerve Agents by a Genetically Engineered Escherichia coli Strain with a Surface-Expressed Cellulose-Binding Domain and Organophosphorus Hydrolase,” App. and Env. Microbiol. 68(4): 1684-1689 (2002), reports degradation of parathion and paraoxon using a strain of E. coli displaying organophosphorus hydrolase (OPH) and a cellulose-binding domain. The OPH was expressed on the cell surface using the Lpp-OmpA fusion system or the truncated ice nucleation protein anchor.
B. Catalyst Supports
There is a need for catalyst supports that are uniform in size and that have the catalyst distributed evenly over the surface of the catalyst support. Uniform supports offer a number of advantages, including minimizing the effects of diffusion and differences between catalyst sites. Catalyst supports may also aid in convenient handling of some catalysts.
One example of generally uniform inorganic catalyst supports may be found in United States Patent Application Publication No. 2006/0009354, to Yueng, et al. The '354 publication reports a catalytic material comprising a metal catalyst anchored to a metal oxide crystal. The metal catalyst is anchored to the surface of the metal oxide by interacting with a hydroxyl group on the surface of the metal oxide.
Catalysts such as those reported in the '354 publication have a number of disadvantages. For example, they may be unsuitable for organic use. Furthermore, they may allow only the use of metal catalysts. Their production may also be expensive, and their construction may make catalyst regeneration difficult. Each support may be limited to a single catalyst.
Embodiments of the invention provide catalysts and catalyst supports that may address one or more of the above disadvantages of the prior art. Methods of construction, use, and regeneration of those catalysts are also provided.